Human vascular to cardiac tissue selectivity of L- and T-type calcium channel antagonists

1 Voltage-operated calcium channel (VOCC) antagonists are effective antihypertensive and antianginal agents but they also depress myocardial contractility. 2 We compared four L-type calcium channel antagonists, felodipine, nifedipine, amlodipine and verapamil and a relatively T-type selective calcium channel antagonist, mibefradil, on human and rat isolated tissue assays to determine their functional vascular to cardiac tissue selectivity (V/C) ratio. 3 The V/C ratio was calculated as the ratio of the IC50 value of the antagonist that reduced (by 50%) submaximally contracted (K+ 62 mM) human small arteries from the aortic vasa vasorum (vascular, V) mounted in a myograph and the IC50 value of the antagonist that reduced (-)-isoprenaline (6 nM) submaximally stimulated human right atrial trabeculae muscle (cardiac, C) mounted in organ chambers. 4 The average pIC(50) Values (-log IC50 M) for the human vascular preparations were felodipine 8.30, nifedipine 7.78, amlodipine 6.64, verapamil 6.26 and mibefradil 6.22. The average pIC(50) values for the cardiac muscle were felodipine 7.21, nifedipine 6.95, verapamil 6.91, amlodipine 5.94, and mibefradil 4.61. 5 The V/C ratio calculated as antilog [pIC(50)V-pIC(50)C] is thus mibefradil 41, felodipine 12, nifedipine 7, amlodipine 5 and verapamil 0.2. 6 In rat small mesenteric arteries the pIC(50) values for the five drugs were similar to the values for human vasa vasorum arteries contracted by K+ 62 mM. However for methoxamine (10 mu M) contraction in the rat arteries the pIC(50) values were lower for felodipine 7.24 and nifedipine 6.23, but similar for verapamil 6.13, amlodipine 6.28 and mibefradil 5.91. 7 In conclusion in the human tissue assays, the putative T-channel antagonist mibefradil shows the highest vascular to cardiac selectivity ratio; some 3 fold higher than the dihydropyridine, felodipine, and some 200 fold more vascular selective than the phenylalkylamine, verapamil. This favourable vascular to cardiac selectivity for mibefradil, from a new chemical class of VOCC antagonist, may be explained by its putative T-channel selectivity.

Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation.

Binding of YY1 and Oct1 to a novel element that downregulates expression of IL-5 in human T cells

Background: IL-5 controls development of eosinophilia and has been shown to be involved in the pathogenesis of allergic diseases. In both atopic and nonatopic asthma, elevated IL-5 has been detected in peripheral blood and the airways. IL-5 is produced mainly by activated T cells, and its expression is regulated at the transcriptional level. Objective: This study focuses on the functional analysis of the human IL-5 (hIL-5) promoter and characterization of eis-regulatory elements and transcription factors involved in the suppression of IL-5 transcription in T cells. Methods: Methods used in this study include DNase I footprint assays, electrophoretic mobility shift assays, and functional analysis by mammalian cell transfection involving deletion analysis and site-directed mutagenesis. Results: We identified 5 protein binding regions (BRs) located within the proximal hIL-5 promoter. Functional analysis indicates that the BRs are involved in control of hIL-5 promoter activity. Two of these regions, BR3 and BR4 located at positions -102 to -73, have not previously been described as regulators of IL-5 expression in T cells. We show that the BR3 sequence contains a novel negative regulatory element located at positions -90 to -79 of the hIL-5 promoter, which binds Oct1, octamer-like, and YY1 nuclear factors. Substitution mutations, which abolished binding of these proteins to the BR3 sequence, significantly increased hIL-5 promoter activity in activated T cells. Conclusion: We suggest that Oct1, YY1, and octamer-like factors binding to the -90/-79 sequence within the proximal IL-5 promoter are involved in suppression of IL-5 transcription in T cells.

Contribution of beta-adrenoceptor subtypes to relaxation of colon and oesophagus and pacemaker activity of ureter in wild-type and beta(3)-adrenoceptor knockout mice

1 The smooth muscle relaxant responses to the mixed beta(3)-, putative beta(4)-adrenoceptor agonist, (-)-CGP 12177 in rat colon are partially resistant to blockade by the beta(3)-adrenoceptor antagonist SR59230A suggesting involvement of beta(3)- and putative beta(4)-adrenoceptors. We now investigated the function of the putative beta(4)-adrenoceptor and other beta-adrenoceptor subtypes in the colon, oesophagus and ureter of wild-type (WT) and beta(3)-adrenoceptor knockout (beta(3)KO) mice. 2 (-)-Noradrenaline and (-)-adrenaline relaxed KCl (30 mM)-precontracted colon mostly through beta(1)-and beta(3)-adrenoceptors to a similar extent and to a minor extent through beta(2)-adrenoceptors. In colon from beta(3)KO mice, (-)-noradrenaline was as potent as in WT mice but the effects were mediated entirely through beta(1)-adrenoceptors. (-)-CGP 12177 relaxed colon from beta(3)KO mice with 2 fold greater potency than in WT mice. The maintenance of potency for (-)-noradrenaline and increase for (-)-CGP 12177 indicate compensatory increases in beta(1)- and putative beta(4)-adrenoceptor function in beta(3)KO mice. 3 In oesophagi precontracted with 1 mu M carbachol, (-)-noradrenaline caused relaxation mainly through beta(1)-and beta(3)-adrenoceptors. (-)-CGP 12177 (2 mu M) relaxed oesophagi from WT by 61.4+/-5.1% and beta(3)KO by 67.3+/-10.1% of the (-)-isoprenaline-evoked relaxation, consistent with mediation through putative beta(4)-adrenoceptors. 4 In ureter, (-)-CGP 12177 (2 mu M) reduced pacemaker activity by 31.1+/-2.3% in WT and 31.3+/-7.5% in beta(3)KO, consistent with mediation through putative beta(4)-adrenoceptors. 5 Relaxation of mouse colon and oesophagus by catecholamines are mediated through beta(1)- and beta(3)- adrenoceptors in WT. The putative beta(4)-adrenoceptor, which presumably is an atypical state of the beta(1)-adrenoceptor, mediates the effects of(-)-CGP 12177 in colon, oesophagus and ureter.

Identification of regions within the third FnIII-like domain of the IL-5R alpha involved in IL-5 interaction

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5R alpha). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5R alpha (hIL-5R alpha) and mouse IL-5R alpha (mIL-5R alpha) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5R alpha but not the mIL-5R alpha. By using chimeras of the mIL-5R alpha and hIL-5R alpha we demonstrate that residues within the first and third FnIII-like domains of mIL-5R alpha are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5R alpha to mIL-5R alpha within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity, These results show that regions of the third FnIII domain of IL-5R alpha are involved in binding, in addition to the regions in domains one and two of the IL-5R alpha that were identified in an earlier study. (C) 2000 Academic Press.

Multiple conformational epitopes are recognized by natural and induced immunity to the E7 protein of human papilloma virus type 16 in man

The reactivity of sera from patients with cervical cancer with the E7 protein of human papilloma virus type 16 (HPV16) was estimated using a novel non-radioactive immunoprecipitation assay and four established protein-and peptide-based immunoassays. Six of 14 sera from patients with cervical cancer and 1 of 10 sera from healthy laboratory staff showed repeated reactivity with E7 in at least one assay. Four of the 7 reactive sera were consistently reactive in more than one assay, but only one was reactive in all four assays. Following immunization with E7, 2 of 5 patients with cervical cancer had increased E7-specific reactivity, measurable in one or more assays. No single assay was particularly sensitive for E7 reactivity, or predictive of cervical cancer. Mapping of E7 reactivity to specific E7 peptides was unsuccessful, suggesting that natural or induced E7 reactivity in human serum is commonly directed to conformational epitopes of E7, These results suggest that each assay employed with is study measures a different aspect of E7 reactivity, and that various reactivities to E7 may manifest following HPV infection or immunization. This finding is of significance for monitoring of E7 immunotherapy and for serological screening for cervical cancer. Copyright (C) 2000 S.Karger, AG. Basel.

Quantitative analysis of vascular to cardiac selectivity of L- and T-type voltage-operated calcium channel antagonists in human tissues

1. Classical L-type voltage-operated calcium channel (VOCC) antagonists dilate blood vessels, depress myocardial contractility and slow cardiac conduction. 2. We compared four L-type VOCC antagonists and a novel tetralol derivative, mibefradil, reportedly 10-fold more selective for T- (transient) over L-type VOCC in two in vitro assays of human tissue, namely isolated small arteries from the aortic vasa vasorum in a myograph and right atrial trabeculae muscle under isometric force conditions. 3. In arteries contracted with K+ (62 mmol/L), the relaxation pIC(50) values for the VOCC antagonists felodipine, nifedipine, amlodipine, verapamil and mibefradil were 8.30, 7.78, 6.64, 6.26 and 6.22, respectively. In atrial trabeculae, the pIC(50) values to inhibit the inotropic response to a submaximal concentration of isoprenaline (6 nmol/L) for felodipine, nifedipine, verapamil, amlodipine and mibefradil were 7.21, 6.95, 6.91, 5.94 and 4.61, respectively. 4. Taking the anti-log (pIC(50) vessel - pIC(50) atrium) the vascular relaxation to cardiac depression potency ratios for mibefradil, felodipine, nifedipine, amlodipine and verapamil were 41, 12, 7, 5 and 0.22, respectively. 5. We conclude that, in human tissue assays, perhaps T- over L-type VOCC selectivity confers the most favourable vascular selectivity on mibefradil. Alternatively, splice variants of L-type VOCC in the vasculature (CaV1.2b) may be more sensitive to mibefradil than the splice variants in the heart (CaV1.2a).

Identification of a minimal promoter sequence for the human N-acetyltransferase Type I gene that binds AP-1 (activator protein 1) and YY-1 (Yin and Yang 1)

Human N-acetyltransferase Type I (NAT1) catalyses the acetylation of many aromatic amine and hydrazine compounds and it has been implicated in the catabolism of folic acid. The enzyme is widely expressed in the body, although there are considerable differences in the level of activity between tissues. A search of the mRNA databases revealed the presence of several NAT1 transcripts in human tissue that appear to be derived from different promoters. Because little is known about NAT1 gene regulation, the present study was undertaken to characterize one of the putative promoter sequences of the NAT1 gene located just upstream of the coding region. We show with reverse-transcriptase PCR that mRNA transcribed from this promoter (Promoter 1) is present in a variety of human cell-lines, but not in quiescent peripheral blood mononuclear cells. Using deletion mutant constructs, we identified a 20 bp sequence located 245 bases upstream of the translation start site which was sufficient for basal NAT1 expression. It comprised an AP-1 (activator protein 1)-binding site, flanked on either side by a TCATT motif. Mutational analysis showed that the AP-1 site and the 3' TCATT sequence were necessary for gene expression, whereas the 5' TCATT appeared to attenuate promoter activity. Electromobility shift assays revealed two specific bands made up by complexes of c-Fos/Fra, c-Jun, YY-1 (Yin and Yang 1) and possibly Oct-1. PMA treatment enhanced expression from the NAT1 promoter via the AP-1-binding site. Furthermore, in peripheral blood mononuclear cells, PMA increased endogenous NAT1 activity and induced mRNA expression from Promoter I, suggesting that it is functional in vivo.

Morphological and electrochemical investigation of RuO(2)-Ta(2)O(5) oxide films prepared by the Pechini-Adams method

Preparation methods can profoundly affect the structural and electrochemical properties of electrocatalytic coatings. In this investigation, RuO(2)-Ta(2)O(5) thin films containing between 10 and 90 at.% Ru were prepared by the Pechini-Adams method. These coatings were electrochemically and physically characterized by cyclic voltammetry, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). The composition and morphology of the oxide were investigated before and after accelerated life tests (ALT) by EDX and SEM. SEM results indicate typical mud-flat-cracking morphology for the majority of the films. High resolution SEMs reveal that pure oxide phases exhibit nanoporosity while binary compositions display a very compact structure. EDX analyses reveal considerable amounts of Ru in the coating even after total deactivation. XRD indicated a rutile-type structure for RuO(2) and orthorhombic structure for Ta(2)O(5). XPS data demonstrate that the binding energy of Ta is affected by Ru addition in the thin films, but the binding energy of Ru is not likewise influenced by Ta. The stability of the electrodes was evaluated by ALT performed at 750 mA cm(-2) in 80 degrees C 0.5 mol dm(-3) H(2)SO(4). The performance of electrodes prepared by the Pechini-Adams method is 100% better than that of electrodes prepared by standard thermal decomposition.

Porphyrin-phospholipid interaction and ring metallation depending on the phospholipid polar head type

The interaction between a hydrophobically modified 5,10,15,20-tetrakis(4-N-tetradecyl-pyridyl) porphyrin and three phospholipids: two negatively charged, DMPA (the sodium salt of dimyristoyl-sn-glycero-phosphatidyl acid) and DMPG (the sodium salt of 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) and a zwitterionic DMPC (dimyristoyl-sn-glycero-phosphatidylcholine), were studied by means of surface pressure isotherms and spectroscopic methods. The interaction results in partial or total metallation of the porphyrin with zinc ions in the presence of negatively charged phospholipids, as attested by UV-vis and luminescence spectroscopy of the transferred films. In the presence of the zwitterionic phospholipid no insertion of zinc ion in the porphyrin ring is detected. These results are relevant for the understanding of photosensitizer-lipid-carrier binding for use in photodynamic therapy. (C) 2010 Elsevier Inc. All rights reserved.

Prospective randomized controlled trial comparing 2 versions of laparoscopic ileal interposition associated with sleeve gastrectomy for patients with type 2 diabetes with BMI 21-34 kg/m(2)

Background: The objective of the present study was to prospectively evaluate the results of 2 versions of laparoscopic ileal interposition (II) and sleeve gastrectomy (SG) for the treatment of patients with type 2 diabetes mellitus and body mass index of 21-34 kg/m(2). Methods: The laparoscopic procedures were prospectively and randomly performed in 38 patients. Of the 38 patients, 18 underwent the first version (II-SG) and 20 underwent the second version in which a diversion of the second portion of the duodenum was applied (II-DSG) and a segment of ileum was interposed into the proximal duodenum. The groups were comparable regarding age (56 and 50 years); gender (13 men and 5 women and 14 men and 6 women); weight (78 and 86 kg); mean BMI (27 and 29 kg/m(2)); duration of type 2 diabetes mellitus (10.1 and 9.2 years); the presence of dyslipidemia (12 and 8 patients), micro- and macroalbuminuria (9 and 9 patients), hypertension (8 and 15 patients), and retinopathy (5 and 8 patients); and the use of antidiabetic medications and the hemoglobin A1c level (8.6% and 8.4%). All patients were followed up for >= 2 years. Results: The mean hospital stay was 3.4 days for the II-SG and 3.5 days for the II-DSG group. No patient required reoperation. All patients in both groups achieved lower levels of hemoglobin A1c. In the II-SG group. the mean hemoglobin A 1c level was 6.35% (range 4.9-8.1). In the II-DSG group, the mean hemoglobin A 1c level was 5.39% (range 4.2-6.5%). The mean BMI decreased in both groups to 22.2 kg/m(2) in the II-SG group and 22.7 kg/m(2) in the II-DSG group. Normal cholesterol levels (<200 mg/dL) were observed in 95% of the II-SG group and 100% of the II-DSG group. The triglycerides were lower than 150 mg/dL in 73% of the II-SG group and 90% of the II-DSG group after 24 months. Conclusion: Laparoscopic II-SG and II-DSG were safe and effective operations for controlling type 2 diabetes mellitus in a nonobese (BMI 21-34 kg/m(2)) population. (Surg Obes Relat Dis 2010;6:296-305.) (C) 2010 American Society for Metabolic and Bariatric Surgery. All rights reserved.

Association Between Increased Platelet P-Selectin Expression and Obesity in Patients With Type 2 Diabetes A BARI 2D (Bypass Angioplasty Revascularization Investigation 2 Diabetes) substudy

OBJECTIVE- To determine whether obesity increases platelet reactivity and thrombin activity in patients with type 2 diabetes plus stable coronary artery disease. RESEARCH DESIGN AND METHODS- We assessed platelet reactivity and markers of thrombin generation and activity in 193 patients from nine clinical sites of the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D). Blood taken at the time of enrollment was used for assay of the concentration of prothrombin fragment 1.2 (PT1.2, released when prothrombin is activated) and fibrinopeptide A (FPA, released when fibrinogen is cleaved). Platelet activation was identified with the use of flow cytometry in response to 0, 0.2, and 1 mu mol/l adenosine diphosphate (ADP). RESULTS- Concentrations of FPA, PT1.2, and platelet activation in the absence of agonist were low. Greater BMI was associated with higher platelet reactivity in response to 1 mu m ADP as assessed by surface expression of P-selectin (r = 0.29, P < 0.0001) but not reflected by the binding of fibrinogen to activated glycoprotein IIb-IIIa. BMI was not associated with concentrations of FPA or PT1.2. Platelet reactivity correlated negatively with A1C (P < 0.04), was not related to the concentration Of triglycerides in blood, and did not correlate with the concentration of C-reactive peptide. CONCLUSIONS- Among patients enrolled in this substudy of BARI 2D, a greater BMI was associated with higher platelet reactivity at the time of enrollment. Our results suggest that obesity and insulin resistance that accompanies obesity may influence platelet reactivity in patients with type 2 diabetes.

Laparoscopic treatment of metabolic syndrome in patients with type 2 diabetes mellitus

Background Metabolic syndrome refers to risk factors for cardiovascular disease. Hyperglycemia is a critical component contributing to the predictive power of the syndrome. This study aimed to evaluate the results from the laparoscopic interposition of an ileum segment into the proximal jejunum for the treatment of metabolic syndrome in patients with type 2 diabetes mellitus and a body mass index (BMI) lower than 35. Methods Laparoscopic procedures were performed for 60 patients (24 women and 36 men) with a mean age of 51.7 +/- 6.4 years (range, 27-66 years) and a mean BMI of 30.1 +/- 2.7 (range, 23.6-34.4). All the patients had a diagnosis of type 2 diabetes mellitus (T2DM) given at least 3 years previously and evidence of stable treatment using oral hypoglycemic agents, insulin, or both for at least 12 months. The mean duration of type 2 diabetes mellitus was 9.6 +/- 4.6 years (range, 3-22 years). Metabolic syndrome was diagnosed for all 60 patients. Arterial hypertension was diagnosed for 70% of the patients (mean number of drugs, 1.6) and hypertriglyceridemia for 70%. High-density lipoprotein was altered in 51.7% of the patients and the abdominal circumference in 68.3%. Two techniques were performed: ileal interposition (II) into the proximal jejunum and sleeve gastrectomy (II-SG) or ileal interposition associated with a diverted sleeve gastrectomy (II-DSG). Results The II-SG procedure was performed for 32 patients and the II-DSG procedure for 28 patients. The mean postoperative follow-up period was 7.4 months (range, 3-19 months). The mean BMI was 23.8 +/- 4.1 kg/m(2), and 52 patients (86.7%) achieved adequate glycemic control. Hypertriglyceridemia was normalized for 81.7% of the patients. An high-density lipoprotein level higher than 40 for the men and higher than 50 for the women was achieved by 90.3% of the patients. The abdominal circumference reached was less than 102 cm for the men and 88 cm for the women. Arterial hypertension was controlled in 90.5% of the patients. For the control of metabolic syndrome, II-DSG was the more effective procedure. Conclusions Laparoscopic II-SG and II-DSG seem to be promising procedures for the control of the metabolic syndrome and type 2 diabetes mellitus. A longer follow-up period is needed.

The 2.0 angstrom crystal structure of a pocilloporin at pH 3.5: The structural basis for the linkage between color transition and halide binding

The pocilloporin Rtms5 and an engineered variant Rtms5(H146S) undergo distinct color transitions (from blue to red to yellow to colorless) in a pH-dependent manner. pK(a) values of 4.1 and 3.2 were determined for the blue (absorption lambda(max), 590 nm) to yellow (absorption lambda(max), similar to 453 nm) transitions of Rtms5 and Rtms5H(146). The pK(a) for the blue-yellow transition of Rtms5H(146S) increased by 1.4 U in the presence of 0.1 M KI, whereas the pK(a) for the same transition of Rtms5 was relatively insensitive to added halides. To understand the structural basis for these observations, we have determined to 2.0 A resolution the crystal structure of a yellow form of Rtms5(H146S) at pH 3.5 in the presence of iodide. Iodide was found occupying a pocket in the structure with a pH of 3.5, forming van der Waals contacts with the tyrosyl moiety of the chromophore. Elsewhere, it was determined that this pocket is occupied by a water molecule in the Rtms5(H141S) structure (pH 8.0) and by the side chain of histidine 146 in the wild-type Rtms5 structure. Collectively, our data provide an explanation for the observed linkage between color transitions for Rtms5(H146S) and binding to halides.

Association between tumoral GH-releasing peptide receptor type 1a mRNA expression and in vivo response to GH-releasing peptide-6 in ACTH-dependent Cushing`s syndrome patients

Objective: GH secretagogues (GHS) produce exaggerated ACTH and cortisol responses in Cushing`s disease (CD) patients, attributable to their direct action on GH-releasing peptide receptor type la (GHSR-1a). However, there are no studies correlating the ill vivo response to GHS and GHSR-1a mRNA expression in ACTH-dependent Cushing`s syndrome (CS) patients. The aim of this study is to correlate the patterns of ACTH and cortisol response to GH-releasing peptide-6 (GHRP-6) to GHSR-1a expression in ACTH-dependent CS patients Design: Prospective study in a tertiary referral hospital center. Fifteen CD patients and two ectopic ACTH syndrome (EAS) patients were studied. Methods: Tumor fragments were submitted to RNA extraction, and GHSR-1a expression was studied through real-time qPCR and compared with normal tissue samples. The patients were also submitted to desmopressin test and vasopressin receptor type 1B (AVPR1B) mRNA analysis by qPCR. Results: GHSR-1a expression was similar in normal pituitary samples and in corticotrophic tumor samples. GHSR-1a expression was higher in patients (CD and EAS) presenting ill vivo response to GHRP-6. Higher expression of AVPR1B was observed in the EAS patients responsive to desmopressin, as well as in corticotrophic tumors, as compared with normal pituitary samples, but no correlation between AVPR1B expression and response to desmopressin was observed in the CD patients. Conclusions: Our results revealed a higher expression of GHSR-1a in the ACTH-dependent CS patients responsive to GHRP-6, suggesting an association between receptor gene expression and ill vivo response to the secretagogue in both the CD and the EAS patients.